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Bacteroidetocins are a family of antibacterial peptide toxins that are produced by and target members of the phylum Bacteroidetes. To date, 19 bacteroidetocins have been identified, and four have been tested and shown to kill diverse Bacteroidales species (M. J. Coyne, N. Bechon, L. M. Matano, V. L. McEneany, et al., Nat Commun 10:3460, 2019, https://doi.org/10.1038/s41467-019-11494-1). Here, we identify the target and likely mechanism of action of the bacteroidetocins. We selected seven spontaneous mutants of four different genera, all resistant to bacteroidetocin A (Bd-A) and found that all contained mutations in a single gene, bamA. Construction of three of these bamA mutants in the wild-type (WT) strains confirmed they confer resistance to Bd-A as well as to other bacteroidetocins. We identified an aspartate residue of BamA at the beginning of exterior loop 3 (eL3) that, when altered, renders strains resistant to Bd-A. Analysis of a panel of diverse Bacteroidales strains showed a correlation between the presence of this aspartate residue and Bd-A sensitivity. Fluorescence microscopy and transmission electron microscopy (TEM) analysis of Bd-A-treated cells showed cellular morphological changes consistent with a BamA defect. Transcriptomic analysis of Bd-A-treated cells revealed gene expression changes indicative of cell envelope stress. Studies in mice revealed that bacteroidetocin-resistant mutants are outcompeted by their WT strain in vivo. Analyses of longitudinal human gut isolates showed that bamA mutations leading to bacteroidetocin resistance do not become fixed in the human gut, even in bacteroidetocin-producing strains and nonproducing coresident strains. Together, these data lend further support to the applicability of the bacteroidetocins as therapeutic peptides in the treatment of maladies involving Bacteroidales species. IMPORTANCE The bacteroidetocins are a newly discovered class of bacteriocins specific to Bacteroidetes with a spectrum of targets extending from symbiotic gut Bacteroides, Parabacteroides, and Prevotella species to pathogenic oral and vaginal Prevotella species. We previously showed that one such bacteroidetocin, Bd-A, is active at nanomolar concentrations, is water soluble, and is bactericidal, all desirable features in a therapeutic antibacterial peptide. Here, we identify the target of several of the bacteroidetocins as the essential outer membrane protein BamA. Although mutations in bamA can be selected in bacteria grown in vitro, we show both in a mouse model and in human gut ecosystems that bamA mutants leading to Bd-A resistance are fitness attenuated and are not selected. These features further support the potential usefulness of the bacteroidetocins as therapeutics for maladies associated with pathogenic Prevotella species, such as recurrent bacterial vaginosis, for which there are few effective treatments.

The selective permeability of the Gram-negative outer membrane (OM) is maintained by integral β-barrel outer membrane proteins (OMPs). The heteropentomeric β-barrel assembly machine (Bam) folds and inserts OMPs into the OM. Coordination of the essential proteins BamA and BamD is critical for OMP assembly and therefore the viability of the cell. The role of the nonessential lipoproteins BamBCE has yet to be characterized; however, genetic evidence suggests that they have nonoverlapping roles in OMP assembly. In this work, we quantify changes of the proteome in the conditional lethal ΔbamB ΔbamE double mutant. We show that cells lacking BamB and BamE have a global OMP defect that is a result of a lethal obstruction of an assembly-competent Bam complex by the lipoprotein RcsF. RcsF is a stress-sensing lipoprotein that is threaded through the lumen of abundant β-barrel OMPs by the Bam complex to expose the amino terminus on the cell surface. We demonstrate that simply removing this lipoprotein corrects the severe OMP assembly defect of the double mutant nearly as efficiently as a previously isolated suppressor mutation in bamA. We propose that BamB and BamE play crucial, nonoverlapping roles to coordinate the activities of BamA and BamD during OMP biogenesis. IMPORTANCE Protein assembly into lipid bilayers is an essential process that ensures the viability of diverse organisms. In Gram-negative bacteria, the heteropentomeric β-barrel assembly machine (Bam) folds and inserts proteins into the outer membrane. Due to its essentiality, outer membrane protein (OMP) assembly by the Bam complex is an attractive target for antibiotic development. Here, we show that the conditional lethal phenotype of a mutant lacking two of the three nonessential lipoproteins, BamB and BamE, is caused by lethal jamming of the stripped-down Bam complex by a normally surface-exposed lipoprotein, RcsF. The heterotrimeric Bam complex (BamA, BamD, BamC) is nearly as efficient as the wild-type complex in OMP assembly if RcsF is removed. Our study highlights the importance of BamB and BamE in regulating the interaction between BamA and BamD and expands our understanding of the role of the Bam complex in outer membrane biogenesis.

The β-barrel assembly machinery, the Bam complex, consists of five components, BamA to -E, among which BamA and BamD are highly conserved and essential. The nonessential components are believed to play redundant roles simply by improving the rate of β-barrel folding. Here we show that BamE contributes a specific and nonoverlapping function to the Bam complex. BamE coordinates BamA and BamD to form a complex between the lipoprotein RcsF and its partner outer membrane β-barrel protein, allowing RcsF to reach the cell surface. In the absence of BamE, RcsF accumulates on BamA, thus blocking the activity of the Bam complex. As the Bam complex is a major antibiotic target in Gram-negative bacteria, the discovery that a lipoprotein can act as a jamming substrate may open the door for development of novel Bam complex inhibitors., The β-barrel assembly machinery, the Bam complex, is central to the biogenesis of integral outer membrane proteins (OMPs) as well as OMP-dependent surface-exposed lipoproteins, such as regulator of capsule synthesis protein F (RcsF). Previous genetic analysis established the model that nonessential components BamE and BamB have overlapping, redundant functions to enhance the kinetics of the highly conserved BamA/BamD core. Here we report that BamE plays a specialized nonredundant role in the Bam complex required for surface exposure of RcsF. We show that the lack of bamE, but not bamB, completely abolishes assembly of RcsF/OMP complexes and establish that the inability to assemble RcsF/OMP complexes is a molecular reason underlying all synthetic lethal interactions of ΔbamE. Our genetic analysis and biochemical cross-linking suggest that RcsF accumulates on BamA when BamA cannot engage with BamD because of its limited availability or the incompatible conformation. The role of BamE is to promote proper coordination of RcsF-bound BamA with BamD to complete OMP assembly around RcsF. We show that in the absence of BamE, RcsF is stalled on BamA, thus blocking its function, and we identify the lipoprotein RcsF as a bona fide jamming substrate of the Bam complex.

SurA is a component of the periplasmic chaperone network that plays a central role in biogenesis of integral outer membrane β-barrel proteins (OMPs) in Escherichia coli. Although SurA contains two well-conserved proline isomerase (PPIase) domains, the contribution of these domains to SurA function is unclear. In the present work, we show that defects in OMP assembly caused by mutation of the β-barrel assembly factors BamA or BamB can be corrected by gain-of-function mutations in SurA that map to the first PPIase domain. These mutations apparently bypass the requirement for a stable interaction between SurA and the Bam complex and enhance SurA chaperone activity in vivo despite destabilization of the protein in vitro. Our findings suggest an autoinhibitory mechanism for regulation of SurA chaperone activity through interdomain interactions involving a PPIase domain. We propose a model in which SurA activity is modulated by an interaction between SurA and the Bam complex that alters the substrate specificity of the chaperone., IMPORTANCE The dominant surA mutations described here alter amino acid residues that are highly conserved in eukaryotic homologs of SurA, including Pin1, the human proline isomerase (PPIase) implicated in Alzheimer’s disease and certain cancers. Consequently, a mechanistic description of SurA function may enhance our understanding of clinically important PPIases and their role(s) in disease. In addition, the virulence of Gram-negative bacterial pathogens, such as Salmonella, Shigella, and Escherichia coli O157:H7, is largely dependent on SurA, making this PPIase/chaperone an attractive antibiotic target. Investigating the function of SurA in outer membrane (OM) biogenesis will be useful in the development of novel therapeutic strategies for the disruption of the OM or the processes that are essential for its assembly.

Bacteria that express contact-dependent growth inhibition (CDI) systems outcompete siblings that lack immunity, suggesting that CDI mediates intercellular competition. To further explore the role of CDI in competition, we determined the target cell range of the CDIEC93 system from Escherichia coli EC93. The CdiAEC93 effector protein recognizes the widely conserved BamA protein as a receptor, yet E. coli EC93 does not inhibit other enterobacterial species. The predicted membrane topology of BamA indicates that three of its extracellular loops vary considerably between species, suggesting that loop heterogeneity may control CDI specificity. Consistent with this hypothesis, other enterobacteria are sensitized to CDIEC93 upon the expression of E. coli bamA and E. coli cells become CDIEC93 resistant when bamA is replaced with alleles from other species. Our data indicate that BamA loops 6 and 7 form the CdiAEC93-binding epitope and their variation between species restricts CDIEC93 target cell selection. Although BamA loops 6 and 7 vary dramatically between species, these regions are identical in hundreds of E. coli strains, suggesting that BamAEcoli and CdiAEC93 play a role in self-nonself discrimination., IMPORTANCE Contact-dependent growth inhibition (CDI) systems are widespread among Gram-negative bacteria, enabling them to bind to neighboring bacterial cells and deliver protein toxins that inhibit cell growth. In this study, we tested the role of CDI in interspecies competition using intestinal isolate Escherichia coli EC93 as an inhibitor cell model. Although E. coli EC93 inhibits different E. coli strains, other bacterial species from the intestine are completely resistant to CDI. We show that resistance is due to small variations in the CDI receptor that prevent other species from being recognized as target cells. CDI receptor interactions thus provide a mechanism by which bacteria can distinguish siblings and other close relatives (self) from more distant relatives or other species of bacteria (nonself). Our results provide a possible means by which antimicrobials could be directed to one or only a few related bacterial pathogens by using a specific receptor “zip code.”